Peptide nucleic acid-targeted mutagenesis of a chromosomal gene in mouse cells.
Peptide nucleic acids (PNAs) can bind to single-stranded DNA by Watson-Crick base pairing and can form triple helices via Hoogsteen bonding to DNA/PNA duplexes. A single dimeric PNA molecule can form a clamp via both double- and triple-helix formation. We designed PNAs to bind as clamps to a site in the supFG1 mutation reporter gene carried within a chromosomally integrated, recoverable lambda phage shuttle vector in mouse fibroblasts. The PNAs were introduced into the cells via permeabilization with streptolysin-O, and cellular uptake was confirmed by fluorescein labeling and fluorescent microscopy. PNAs specific for either an 8- or a 10-bp site in the supFG1 gene were found to induce mutations at frequencies in the range of 0.1%, 10-fold above the background. PNAs with three or four mismatches showed poor in vitro target site binding and were ineffective in the mutagenesis assay. No increased mutagenesis was detected with any of the PNAs in the nontargeted cII gene, also carried within the lambda vector, further indicating the specificity of the PNA-induced mutagenesis. DNA sequence analysis revealed that the majority of the mutations were located within the PNA-binding site and consisted mostly of single base pair insertions and deletions within the poly G:C run there, suggesting that a high affinity PNA clamp constitutes a mutagenic lesion that may provoke replication slippage errors. The ability to direct mutations to a target site in chromosomal DNA by using PNAs may provide a useful tool for research and therapeutic applications.
Faruqi
A F
AF
Departments of Therapeutic Radiology and Genetics, Yale University School of Medicine, P.O. Box 208040, New Haven, CT 06520-8040, USA.
Egholm
M
M
Glazer
P M
PM
D000818:Animals; D010582:Bacteriophage lambda; D002460:Cell Line; D002875:Chromosomes / Q000187:drug effects / Q000648:ultrastructure; D004247:DNA / Q000235:genetics*; D016153:Genes, Suppressor; D006860:Hydrogen Bonding; D051379:Mice; D008822:Mice, Transgenic; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D010455:Peptides
D009838:Oligodeoxyribonucleotides; D010455:Peptides; D004247:DNA